Anaplastic lymphoma kinase (ALK) has been identified as the fusion partner of echinoderm microtubule-associated protein-like 4 (EML4) in non–small-cell lung cancer (NSCLC), and this fusion accounts for 3.0 to 6.7% of NSCLC patients.1,2 Despite various fusion variants2 and partners, including TRK-fused gene3 and kinesin family member 5B (KIF5B),4,5 the complete intracellular portion of ALK is preserved and oncogenic. In vitro and in vivo sensitivities to ALK tyrosine kinase inhibitors6 were translated into clinical activities of crizotinib, a dual inhibitor of ALK and c-MET against ALK-positive NSCLC.7 A recent phase I trial of crizotinib resulted in an objective response rate of 57%, a disease control rate of 87% at 8 weeks, and a median progression-free survival of 10 months.7,8 In addition, a first-line phase III trial comparing crizotinib and pemetrexed plus cisplatin is currently ongoing in ALK-rearranged NSCLC patients (NCT01154140). Although ALK-rearranged NSCLC was shown to be highly susceptible to crizotinib treatment,7 a fraction of tumors eventually became resistant to crizotinib.9 In line with the T790M “gatekeeper” mutation in epidermal growth factor receptor (EGFR)-mutant NSCLC,10 L1196M in the ALK gatekeeper site was identified in a Japanese patient who showed acquired resistance to crizotinib.9 In addition, nongatekeeper mutations such as L1152R,11 C1156Y,9 and G1269A,12 and alternative pathway activation, including ligand-dependent EGFR activation,11 EGFR mutation,12 or amplification,13 were suggested to be resistance mechanisms in ALK-positive NSCLC treated with ALK inhibitors.
Here, we evaluated heterogeneous genetic changes associated with crizotinib resistance in seven ALK-rearranged NSCLC patients who initially responded and eventually showed resistance to crizotinib.
PATIENTS AND METHODS
Crizotinib at a dosage of 250 mg twice daily was administered to seven patients with ALK-rearranged NSCLC, of whom four were enrolled into phase I (NCT00585195) trial7 and two were enrolled in phase II (NCT00932451) trial. All were diagnosed with stage IV NSCLC disease, and most patients received crizotinib as second-line treatment and beyond (Table 1). Response evaluation was based on the Response Evaluation Criteria in Solid Tumors version 1.0 per protocol.14 Tumor tissues were obtained in patients who failed crizotinib and provided informed consent. This study was approved by the Institutional Review Board of Seoul National University Hospital (H-1205-126-411).
Molecular Determinations of ALK, EGFR, and KRAS and Measurement of EGFR Ligand Levels
ALK fluorescence in situ hybridization (FISH) was analyzed using an ALK probe (Vysis LSI ALK dual-color, break-apart rearrangement probe; Abbott Molecular, Des Plaines, IL) in formalin-fixed and paraffin-embedded tissues or cell pellets derived from malignant pleural effusions. ALK FISH was considered positive when break-apart signals were observed in more than 15% of 50 or more analyzed cells.7,15 In addition, the number of copies per cell of the ALK gene rearrangement was determined before and after crizotinib, and ALK gene amplification was defined as a greater than twofold increase of the mean of the rearranged gene per cell in the post-crizotinib specimen compared with the precrizotinib specimen.12 EGFR amplification was evaluated using an EGFR probe (Vysis LSI EGFR; Abbott Molecular) and EGFR FISH positivity was determined according to the Colorado scoring criteria.16 EGFR and KRAS mutations were analyzed using direct sequencing of extracted DNA from NSCLC patient tumors or cell lines.17
Amphiregulin and EGF levels were measured in the cell culture medium or supernatant fluid of pleural effusion using an enzyme-linked immunosorbent assay kit (Quantikine R&D Systems, Minneapolise, MN). All samples were run in at least duplicate. The concentrations of the EGFR ligands were determined by comparison with standard curves.
Cell Lines and Reagents
NCI-H3122 cells were provided by Pasi A. Jänne (Dana-Farber Cancer Institute, Boston, MA) and NCI-H2228 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA). SNU-2535 cells were established at the Korean Cell Bank using pleural effusion in a patient who had developed acquired resistance to crizotinib (SNU4 patient, Table 1). NCI-H3122, NCI-H2228, and SNU-2535 cells were maintained in RPMI 1640 medium with gentamicin (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (GIBCO). The cell lines were incubated at 37°C in a humidified atmosphere of 5% carbon dioxide. Crizotinib was kindly provided by Pfizer (Milwaukee, WI) and dissolved in dimethyl sulfoxide (Sigma Aldrich, St. Louis, MO) for experiments. Total ALK (#3333), phosphorylated ALK Tyr 1604 (#3341), total AKT (#4685), phosphorylated AKT (#4060S), total ERK p42/44 (#9102), phosphorylated ERK (#9106), and GAPDH (#5174) antibodies were purchased from Cell Signaling Technology (Danvers, MA) and were used for immunoblotting. The blots were washed, transferred to freshly prepared enhanced Lumi-Light Western Blotting Substrate (Roche, Indianapolis, IN), and subjected to imaging analysis using an LAS-3000 imaging system (Fuji Photo Film Co., Stamford, CT).
Reverse Transcription–Polymerase Chain Reaction and Genotyping
Total RNA was isolated from the NSCLC cell lines using an RNA Mini kit (Invitrogen, Carlsbad, CA). Each sample was reverse transcribed into cDNA using SuperScript II reverse transcriptase (Invitrogen). PCR amplification was performed using protocols with specific primers (Supplementary Table 1, Supplemental Digital Content 1, http://links.lww.com/JTO/A387). The images were captured using the Gel Logic 200 imaging system (Kodak, Rochester, NY). The PCR products were purified using a PCR purification kit (Invitrogen) and a DNA concentration kit (ZYMO RESEARCH, Irvine, CA) and subsequently sequenced as described later. Genomic DNA was isolated from cell pellets or tissues using an ALL-prep DNA/RNA micro kit (QIAGEN, Valencia, CA) according to the manufacturer’s protocol. Exons of ALK were amplified from genomic DNA using the High Fidelity plus PCR system (Roche, Indianapolis, IN) and sequenced bi-directionally by Sanger dideoxynucleotide sequencing with the primers for ALK exons 23 and 25 (Supplementary Table 1, Supplemental Digital Content 1, http://links.lww.com/JTO/A387). Mutation-specific PCR of L1196M and G1269A were performed with primer sequences. Direct sequence analysis was performed using an ABI 3730 DNA sequencer (Applied Biosystems, Carlsbad, CA). The reference sequences for ALK and EML4 were NM_004304.4 and NM_019063.3, respectively. All mutations were confirmed at least twice from independent PCR isolates and sequence tracing was reviewed in the reverse direction by visual inspection.
Cloning of EML4-ALK Expression Constructs
EML4-ALK variant 1 cDNA from H3122 cells was amplified using primers. The PCR products were cloned into the TOPOTA vector (Invitrogen) and transformed into bacteria, and the ALK kinase domain (residues 1094–1396) was sequenced in individual clones. To generate EML4-ALK mutants, the L1196M or G1269A mutation was introduced using site-directed mutagenesis (Agilent Technologies, La Jolla, CA) with mutant-specific primers according to the manufacturer’s instructions. The PCR primers are listed in Supplementary Table 1 (Supplemental Digital Content 1, http://links.lww.com/JTO/A387). The sequencing products were analyzed using Sequencer software (Gene codes). The Basic Local Alignment and Search Tool (BLAST database) was used to determine the identification of unknown sequences. Retroviral infection and culture of Ba/F3 cells were performed using a previously described method.1 Polyclonal cell lines were established by puromycin selection and subsequently cultured in the absence of interleukin-3. Uninfected Ba/F3 cells were used as controls.
Cell Proliferation and Apoptosis Assays
NSCLC cells at a density of 5000 cells per well in 96-well plates were cultured in the presence of crizotinib or vehicle for 72 hours, and cell proliferation was analyzed using the CCK-8 colorimetric assay (Dojindo, Tokyo, Japan). The absorbance was measured at 450 nm in an Eon Microplate Spectrophotometer (BioTek, Winoosk, VT) using at least duplicate samples. The NSCLC cells treated with crizotinib or vehicle were stained with Annexin V and 7 AAD, and then the cells were analyzed by flow cytometry using a FACSCalibur instrument (Becton Dickinson, Franklin Lakes, NJ).
Patient Outcomes and the ALK Fusion Transcript Analysis
Treatment outcomes are described in Table 1. All patients achieved a partial response and subsequently experienced disease progression at a median time of 6 months (range, 4–12 months). Although most patients discontinued crizotinib at the time of documented progressive disease, SNU2 patients received crizotinib beyond disease progression owing to ongoing clinical benefit. EML4-ALK fusion transcripts were analyzed by reverse transcription–polymerase chain reaction and PCR fragments that represented variable truncations of EML4 at exons 13 to 20 (432 base pairs) and 6 to 20 (917 base pairs) were sequenced. Variant 1 that fuses EML4 exon 13 to ALK exon 20 (E13;A20) was identified in four patients and variant 3 (E6;A20) was observed in two patients (Table 2 and Fig. 1A). One patient harbored an ALK fusion at exon 20 with exon 15 of KIF5B.
ALK Mutations in Crizotinib-Resistant Tumors
The G→C and C→A substitutions at nucleotides 4713 (G1269A) and 4493 (L1196M) of ALK were identified and confirmed by Sanger sequencing. Of note, the SNU1 tumor harbored both L1196M (frequency, 6.59%) and G1269A (28.5%) mutations in separate clones (Fig. 1B). The SNU4 patient displayed the G1269A mutation at a frequency of 68.75%. In addition, the L1196M mutation was found in SNU6 tumoral cDNA clones (27.8%). These were confirmed using L1196M or G1269A mutation-specific PCR (Fig. 1C). However, the remaining four patients demonstrated no secondary mutation within the ALK kinase domain. SNU-2535 derived from an SNU4 patient harbored the G1269A mutation (61.9%).
ALK Mutations Confer Resistance to Crizotinib
293T cells were transiently transfected with cDNA encoding wild-type (WT) EML4-ALK, EML4-ALK L1196M, or EML4-ALK G1269A to evaluate resistant properties of these mutations. ALK phosphorylation was less decreased in 293T-L1196M or -G1269A cells than in 293T-WT cells after exposure to crizotinib in a dose-dependent manner (Fig. 2A). In addition, Ba/F3 cells that expressed secondary ALK mutations were resistant to crizotinib treatment compared with those expressing WT EML4-ALK (Fig. 2B). Apoptotic cells determined by Annexin V and 7-AAD were higher in H3122 cells than in SNU-2353 cells treated with crizotinib (Fig. 2C). H3122 CR1 cells that were generated by an increasing dose of crizotinib to 1 μM13 and SNU-2535 cells were resistant to crizotinib compared with H3122 cells (half maximal inhibitory concentration [IC50], 6.01 and 4.1 µM, respectively; Fig. 3A). Crizotinib treatment suppressed expression of phospho-ALK, -AKT, and -ERK in H3122 cells. However, crizotinib ineffectively suppressed phosphorylated proteins in SNU-2535 and H3122 CR1 cells (Fig. 3B). Apoptotic cells were reduced in SNU-2535 cells compared with those in H3122 cells after exposure to crizotinib (Fig. 3C).
Heterogeneous Genetic Changes in Crizotinib-Resistant Tumors
Although patients with secondary ALK mutations showed no other genetic alteration, those without these mutations were tested for genetic alterations of alternative pathways (Table 2). Of note, the SNU5 patient had an ALK gene amplification (ALK gene copy number gain, 4.1-fold increase compared with the precrizotinib specimen; Fig. 4A) and L858R mutation in EGFR exon 21 with EGFR high polysomy (EGFR/CEP = 1.79; Fig. 4B). She carried a WT EGFR gene with disomy in the initial tumor tissue.
However, genetic alterations associated with acquired resistance to crizotinib were not found in three ALK-positive NSCLC patients. In addition, ALK or EGFR gene amplification and EGFR or KRAS mutation were not observed in SNU-2535 cells. KRAS gene mutation was not found in all tumor tissues.
The amphiregulin concentration was significantly high in supernatant fluid from SNU1 (1894.0 pg/ml) and SNU6 (1832.5 pg/ml) patients than the amphiregulin concentration in supernatant fluid from SNU2, 4, and 7 patients (116.4–460.0 pg/ml; Fig. 4C). Similarly, SNU1 and SNU6 patients demonstrated relatively high EGF concentrations (Fig. 4D). EGFR ligand levels were significantly high in SNU-2535 and H3122 CR1 cells than in H3122 cells.
Our study demonstrates that acquired resistance mechanisms are heterogeneous in ALK-rearranged NSCLC treated with crizotinib. Three of seven patients (43%) had secondary ALK mutations and one patient (14%) showed a high ALK gene copy number and activation of EGFR signals expressed by high EGFR polysomy and L858R in EGFR exon 21. In addition, five patients with malignant pleural effusions showed a high concentration of amphiregulin.
L1196M is a “gatekeeper” mutation in ALK, which is analogous to T315I in ABL and T790M in EGFR, and sterically interferes with crizotinib binding.9 This confers a high level of resistance to crizotinib with an IC50 of 1000 nM or more and is identified even at the highest dose of crizotinib in in vitro mutagenesis screening.7,18 Recently, six patients with ALK-rearranged NSCLC who received crizotinib harbored L1196M mutation after a median duration of 6 months (range, 4–28 months), including two patients in this study.9,12,19 The insensitivity of L1196M clones to crizotinib and ineffective suppression of phospho-ALK was confirmed in our Ba/F3 and 293T systems, similar to previous studies.9,11–13,19 Mutation at position G1269 in the DFG motif was identified by in vitro mutagenesis18 and in two patients who showed acquired resistance to crizotinib.12 G1269A mediated an intermediate resistance between C1156Y and L1196M,12 a finding that was similarly observed in our analysis. Currently, four patients with ALK-positive NSCLC received crizotinib for a median duration of 9 months (range, 6–13 months) until disease progression with G1269A acquisition.12 Of note, one patient had both L1196M and G1269A mutations that were confirmed by a highly sensitive mutation-specific PCR assay in different clones, findings similar to those in a case report,9 suggesting heterogeneous tumor clones under a different pressure within the same patient. We found no ALK secondary mutation such as 1151Tins,19 L1152R,11 C1156Y,9 F1174C,20 G1202R,19 D1203N,20 or S1206Y19 in our patients. However, because of unknown results of L1196M and G1269A in tumors before crizotinib, we do not know truly gained secondary ALK mutations in SNU1, 4, and 6 patients.
ALK copy number gain was observed in partially resistant H3122 cells, resulting in a step-wise evolution of acquired resistance to crizotinib.13 This was confirmed in three patients, of whom one had a G1269A mutation,12,19 and in our patient. Of note, our patient with ALK gene amplification harbored EGFR mutation (L858R) and EGFR high polysomy. She had no detectable EGFR mutation by direct sequencing before crizotinib and her tumor responded to crizotinib only for 4 months. Thus far, two patients have co-existent ALK dominant and nondominant mechanisms of resistance: one patient harbors G1202R mutation and KIT amplification,19 whereas the other patient displayed ALK copy number gain and EGFR mutation with high polysomy. These findings suggest heterogeneous resistance mechanisms associated with crizotinib treatment even in the same patient with ALK-rearranged NSCLC.
ALK nondominant mechanisms of resistance consist of second oncogene activation with partial ALK dependence and separate oncogene activation with ALK independence.20 The former includes ligand-dependent EGFR11 or KIT19 activation; and the latter includes KRAS or EGFR mutation.12 Of note, five patients with malignant pleural effusions at the time of crizotinib resistance had high amphiregulin concentration in supernatant fluid, suggesting ligand-dependent EGFR activation. However, unknown results of EGFR ligands in pre-crizotinib samples and EGFR phosphorylation in tumors might weaken the possible resistance mechanism associated with EGFR signals.
In conclusion, genetic changes associated with acquired crizotinib resistance are heterogeneous in ALK-rearranged NSCLC in our study. Despite a small number of patients and preexistent similar studies, it is hoped that demonstrated high concentrations of EGFR ligands in patients’ samples and identified one patient with both ALK dominant and nondominant mechanisms of crizotinib resistance. Although we successfully identified ALK dominant and nondominant resistance mechanisms in four (57%) and one (14%) of seven patients, respectively, these were not observed in three patients. Therefore, a new technique such as next-generation sequencing is necessary to uncover a novel mechanism associated with crizotinib resistance. In addition, identification of resistant mechanisms might help to select a patient who benefits from new ALK-directed therapies: new agents such as an HSP90 inhibitor, or a combination of crizotinib and a new agent.
The authors thank Su Jung Huh and Jieun Yoon for assistance with tumor sample collection and in vitro experiments. This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (A110057).
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Anaplastic lymphoma kinase; Non–small-cell lung cancer; Crizotinib; Acquired resistance; ALK mutation